The present invention relates to a technique for treating a condition or disease in a mammal, including humans, related to supranormal intracellular enzyme activity, and to a prodrug useful in treating such a condition or disease.
Many of the most prevalent diseases in humans including ischemia, stroke, epilepsy, asthma and allergy are all believed to be related to the phenomenon of cell hyperexcitation, a term used herein to denote supranormal intracellular enzyme activity. Certain pharmacological strategies are therefore aimed at inhibiting this detrimental degradative activity.
In contrast to such known strategies which are aimed at suppressing this degradative activity, it would be advantageous to be able to selectively target diseased cells characterized by enzyme hyperactivity, so as to introduce a pharmacologically active molecule in the form of a prodrug into the cell, whereby such hyperactivity would act on the prodrug, so that the pharmacologically active molecule accumulates in the diseased cells rather than in the healthy cells.
Different types of intracellular enzyme systems are known to be significantly elevated in pathological conditions, and may be used to achieve preferential release of the active drug compound within the diseased cells. Candidate enzymes that could be utilized to activate the prodrugs according to the present invention include lipases, proteases or glycosidases. By way of example, in many diseases cell membranes are broken down due to abnormal intracellular lipase activity.
The use of prodrugs to impart desired characteristics such as increased bioavailability or increased site-specificity on known drugs is a recognized concept in the state of the art of pharmaceutical development. The use of various lipids in the preparation of particular types of prodrugs is also known in the background art. In none of those instances are the prodrugs characterized in that they achieve preferential accumulation of the drug within the diseased cells of the organ, by activation with intracellular lipases. Rather, they provide for the drug to be transported to a specific site, or to be released within a specific organ.
This approach is exemplified in the case of the phospholipid prodrugs of salicylates and non-steroidal anti-inflammatory drugs disclosed in WO 91/16920 which, taken orally, protect the gastric mucosa and release the active principle in the gut.
In other examples of phospholipid prodrugs, formulation of the prodrugs into liposomes or other micellar structures is the feature that enables their preferential uptake, for instance by macrophages or by liver cells as in the case of the phospholipid conjugates of antiviral drugs disclosed in WO 90/00555 and WO 93/00910.
Generally, viral infection is not associated with supranormal phospholipase activity and antiviral phospholipid conjugates do not teach or suggest activation of the drug preferentially in the diseased cells, or in the infected cells as in the case of the phospholipid conjugates of antiviral nucleotides and anti-sense oligonucleotides, such as those disclosed in WO 90/00555, in WO 90/10448 and in NTIS Technical Notes, no. 9, page 630, Springfield, Va., US, 1984.
In other instances specific types of polar lipids are used to target the prodrugs to intracellular organelles as in the case of the antiviral and antineoplastic nucleosides disclosed in U.S. Pat. No. 5,149,794. Additional types of lipids have also been used in specific types of prodrugs such as EP A-325160 which discloses glycerin esters of ACE inhibitors, which form micelles absorbed from the intestine into the lymphatic system, thereby bypassing the liver and having increased access to the central nervous system, for use in the treatment of hypertension and cognitive dysfunction. The ACE inhibitors undergo enzymatic cleavage and exert their therapeutic effects extracellularly.
Other types of lipophilic carriers that facilitate intracellular transport are known in the art, as in CH A-679856 which discloses the use of salicyloyl-carnitine for the treatment of pain, and in WO 89/05358 which discloses modified oligonucleotide antisense drugs, transported into cells by attachment of apolar groups such as phenyl or naphthyl groups.
Different classes of pharmacologically active molecules can be administered as prodrugs according to the principles of the present invention. Candidates include anti-inflammatory drugs, anti-epileptic drugs, protease inhibitors, and anti-tumor drugs. A non-limiting example of such pharmacologically active molecules is a calcium chelating agent, which would have many advantages over drugs presently used for the treatment of calcium associated disorders.
Intracellular calcium is an important determinant for cell death, irrespective of the initial insult sustained by the cell. It may be involved in cell death in lymphocyte and killer cell mediated damage of target cells, in organ damage during transplantation, and in other types of tissue damage including ischemic insults. Calcium channel blockers or cell membrane permeable forms of calcium chelators have been suggested to protect against tissue injury or to decrease tissue damage. Thus, it will be apparent that the present invention has potential use (in the embodiment employing a calcium chelator ) in relation to these circumstances
The cell damage occurring in ischemia may be secondary to the influx and/or intracellular release of Ca2+ ions (Choi, Trends Neurosci., 1988, 11, 465-469; Siesjo and Smith, Arzneimittelforschung, 1991, 41, 288-292). Similarly, calcium influx appears to play an important role in the genesis of epileptic seizures. Although a significant portion of intracellular calcium arrives from intracellular stores, current research suggests that calcium entry blockers may have anticonvulsant activity (see e.g. Meyer, 1989, Brain Res. Rev. 14, 227-243).
Drugs which are currently or potentially useful for treatment of calcium associated disorders include: (1) calcium channel blockers, (2) drugs affecting calcium balance by modification of intracellular calcium storage sites, and (3) intracellular calcium chelating agents. Calcium channel blockers used in clinical practice are represented by Verapamil, Nifedipine and Diltiazem. The major toxicities associated with the use of such compounds involve excessive vasodilation, negative inotropy, depression of the sinus nodal rate, and atrial ventricular (A-V) nodal conduction disturbances. Drugs affecting calcium mobilization and/or sequestration, like calcium channel blockers, exhibit rather narrow specificity.
Though the use of calcium chelators for reducing injury to mammalian cells is disclosed in WO 94/08573, there are no intracellular calcium chelating agents suitable for clinical requirements. Existing cell membrane permeable calcium chelators include acetoxymethyl esters such as EGTA-AM (ethylene-1,2-diol bis 2-aminoethyl ether N,N,Nxe2x80x2,Nxe2x80x2-tetra-acetic acid acetoxymethyl ester) EDTA-AM (ethylene-1,2-diamine tetra-acetic acid acetoxymethyl ester), and BAPTA-AM (1,2-bis 2-aminophenoxy ethan-N,N,Nxe2x80x2,Nxe2x80x2-tetra-acetic acid acetoxymethyl ester). These known complex molecules, are digested by ubiquitous esterases, thus causing activation of the chelator in the intracellular space in a manner which is random and uncontrolled, being unrelated to cell activity.
It will also be self-evident that a similar concept can be applied to the treatment of conditions or diseases other than those related to the intracellular level of Ca2+ ions. By way of example, if the active entity incorporated in the prodrug molecule is a protein kinase inhibitor, after administration of the prodrug the inhibitor would be accumulated in a cell exhibiting abnormal proliferation, thus providing potentially an important tool for use in antitumor therapy.
In accordance with one object of the invention, there are provided prodrugs which selectively undergo activation to release pharmacologically active compounds in hyperactivated cells. In accordance with another object of the invention, the pharmacologically active compound is released from the prodrug in response to enzyme activity in the targeted cells. In accordance with yet another object of the invention, the pharmacologically active compound, selectively accumulated in a cell characterized by a relatively raised level of enzyme activity therein, is trapped in the cell and therefore exhibits an enhanced desired activity therein.
The present invention accordingly provides in one aspect, a prodrug which is a covalent conjugate of a pharmacologically active compound and an intracellular transporting adjuvant, characterized by the presence of a covalent bond which is scission-sensitive to intracellular enzyme activity.
In another aspect, the present invention provides a technique for treating a condition or disease in a mammal, including a human, related to supranormal intracellular enzyme activity, which comprises administering to a mammal having such condition or disease, a pharmaceutically acceptable cell membrane permeable prodrug, the prodrug being a covalent conjugate of a pharmacologically active compound and an intracellular transporting adjuvant, characterized by the presence of a covalent bond which is scission-sensitive to intracellular enzyme activity, such that the bond is broken in response to such activity, whereby the pharmacologically active compound accumulates selectively within cells having supranormal intracellular enzyme activity, or in their immediate environment. In one particular aspect, the technique or method is used to treat, e.g., a human patient.
In yet another aspect, the invention provides pharmaceutical compounds for treating a condition or disease in a mammal related to supranormal intracellular enzyme activity, by selectively accumulating a pharmacologically active compound within cells having such activity, comprising a pharmaceutically acceptable cell membrane permeable prodrug, which is a covalent conjugate of the pharmacologically active compound and an intracellular transporting adjuvant, and is characterized by the presence of a covalent bond which is scission-sensitive to intracellular enzyme activity, such that the bond is broken in response to such activity. In one particular aspect, the pharmaceutical compounds are used to treat, e.g., a human patient.